The long-term goal of this project is to understand the role of CYP2E1, ALDH2, and ALDH1 genes which encode alcohol-metabolizing enzymes, in alcohol-induced oxidative stress and toxicity. Chronic alcohol abuse results in a variety of pathological effects, including liver fibrosis and cirrhosis. Some of the toxic effects of ethanol have now been linked to ethanol metabolism. Ethanol consumption causes a CYP2E1 induction, which is associated with increased levels of acetaldehyde, generation of reactive oxygen species (ROS), lipid peroxidation, mitochondrial GSH depletion, and increased collagen synthesis. Most of the toxic effects of ethanol are due to acetaldehyde and potential molecular targets include proteins, lipids and mitochondrial DNA. Genetic polymorphisms exist in human CYP2E1 and ALDH2 genes that may influence the risk for developing alcoholic liver disease. Interestingly, chronic ethanol consumption mimics the oxidative stress seen in aging animals, particularly in liver. Although the importance of oxidative stress in cellular damage is well appreciated, the molecular mechanisms by which alcohol induces oxidative stress and causes liver damage are still unknown. Our hypothesis predicts that chronic alcohol consumption induces oxidative stress and cellular injury that is associated with imbalances in the production/detoxification of intermediates (acetaldehyde) or reactants (ROS and lipid peroxidative products) of ethanol metabolism. To test this hypothesis, the role of Cyp2e1, Aldh2 and Aldh1 genes in ethanol metabolism and liver in injury will be investigated by using transgenic mouse lines having homozygous disruptions in these genes. For the 5-year period of this grant application, we therefore propose to: Specific Aim 1: Develop conventional, as well as inducible, Aldh2 and Aldh1 knockout transgenic mouse lines; Specific Aim 2: Use the Aldh2, Aldh1 and a Cyp2e1 knockout mouse lines to study the role of these genes in ethanol/acetaldehyde pharmacokinetics and metabolism; Specific Aim 3: Use the Cyp2e1 Aldh2 and Aldh1 knockout mouse lines to test the hypothesis that CYP2E1 (involved in ROS production), ALDH2 and ALDH1 I (involved in acetaldehyde detoxification) are involved in the alcohol-induced oxidative stress that leads to liver injury. Liver injury will be studied in parallel with: i) oxidative stress, ii) assessment of lipid peroxidation, iii) characterization of molecular targets of oxidative stress-mediated injury including mitochondrial DNA damage and genes involved in collagen synthesis and degradation. These studies will greatly enhance our understanding of genetic factors involved in alcohol toxicity.